Multisite protein phosphorylation and dephosphorylation (P&D) are fundamental to most eukaryotic cellular processes. Disregulation of multisite P&D is implicated in several human diseases, including cancer, and both kinases and phosphatases are targets of important drugs in clinical use or development. The goal of this proposal is to investigate the dynamical complexity that arises when kinases and phosphatases collectively regulate the cellular phosphorylation state. A substrate molecule with n phosphorylation sites may occupy 2n states and a population of substrate molecules will have a distribution of such phospho-forms, with the system of kinase, phosphatase and substrate being maintained away from equilibrium by a steady supply of ATP. We will develop a mass spectrometry protocol to distinguish all 2n phospho-forms and assemble a set of model P&D systems, having varying numbers of phosphorylation sites, enzymatic mechanisms and cellular roles, on which to undertake systems biochemistry. We will determine mathematically the conditions under which such systems exhibit complex behaviors, such as multistability and oscillations, and will test these predictions on the model systems. Such a multidisciplinary approach will provide novel insights into these complex, medically important and poorly-understood systems. PUBLIC HEALTH RELEVANCE: Disregulation of protein phosphorylation and dephosphorylation is implicated in several human diseases, including cancer, and the enzymes involved, kinases and phosphatases, are targets of important drugs in clinical use and development. Systems of kinase, phosphatase and substrate show surprising complexity, especially as the number of phosphorylation sites increases. This proposal seeks to investigate such complexity through a multidisciplinary mathematical and experimental approach, using several model phosphorylation and dephosphorylation systems having varying cellular roles, enzymatic mechanisms and numbers of phosphorylation sites.